HSATII RNA depletion nor KDM2A depletion does not rescue global H2AK119Ub signal. (A) Quantitative RT-PCR of gapmer-mediated HSATII RNA depletion in cells using gapmers targeting either HSATII or control sequences (Shadle et al., 2019) in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. HSATII transcripts are normalized to RPL27 internal control. N = 3. (B) Paired samples from A for immunofluorescence analysis of RNF2 signal (red) and H2AK119Ub signal (green) in −dox or +dox iDUX4 cells treated with HSATII− or control gapmers. Images represent one experiment. (C) Nuclear H2AUb signal intensity was calculated for nuclei that contained RNF2 pan-nuclear signal (pan) or RNF2 aggregates (foci) in HSATII or control gapmer treated cells. Nuclei are indicated for each independent experiment and N ≥ 50 nuclei per condition. (D) siRNA depletion of either KDM2A (siKDM2A) or control (siCTRL) sequences in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Western blot was performed on whole-cell lysate and probed for KDM2A. GAPDH was used as loading control. Numbers above KDM2A represent signal relative to GAPDH. Blot is representative of three biological replicates per condition. (E) Paired samples from A for combined RNA-FISH and immunofluorescence of HSATII RNA signal (green) and H2AK119Ub signal (red) in +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells with (siKDM2A) or without (siCTRL) KDM2A depletion. Images represent one experiment. (F) Nuclear H2AUb MFI was calculated in +dox iDUX4 cells with (siKDM2A) or without (siCTRL) KDM2A depletion in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+). Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition. (A, C, and F) Data represent means ± SD. Statistical differences between groups were analyzed with one-way ANOVA Tukey’s multiple comparison test or two-way ANOVA Sidak’s multiple comparison test or were assessed with Kruskal–Wallis test between each group and a control. Source data are available for this figure: SourceData FS2.
Figure S2.

HSATII RNA depletion nor KDM2A depletion does not rescue global H2AK119Ub signal. (A) Quantitative RT-PCR of gapmer-mediated HSATII RNA depletion in cells using gapmers targeting either HSATII or control sequences (Shadle et al., 2019) in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. HSATII transcripts are normalized to RPL27 internal control. N = 3. (B) Paired samples from A for immunofluorescence analysis of RNF2 signal (red) and H2AK119Ub signal (green) in −dox or +dox iDUX4 cells treated with HSATII− or control gapmers. Images represent one experiment. (C) Nuclear H2AUb signal intensity was calculated for nuclei that contained RNF2 pan-nuclear signal (pan) or RNF2 aggregates (foci) in HSATII or control gapmer treated cells. Nuclei are indicated for each independent experiment and N ≥ 50 nuclei per condition. (D) siRNA depletion of either KDM2A (siKDM2A) or control (siCTRL) sequences in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Western blot was performed on whole-cell lysate and probed for KDM2A. GAPDH was used as loading control. Numbers above KDM2A represent signal relative to GAPDH. Blot is representative of three biological replicates per condition. (E) Paired samples from A for combined RNA-FISH and immunofluorescence of HSATII RNA signal (green) and H2AK119Ub signal (red) in +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells with (siKDM2A) or without (siCTRL) KDM2A depletion. Images represent one experiment. (F) Nuclear H2AUb MFI was calculated in +dox iDUX4 cells with (siKDM2A) or without (siCTRL) KDM2A depletion in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+). Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition. (A, C, and F) Data represent means ± SD. Statistical differences between groups were analyzed with one-way ANOVA Tukey’s multiple comparison test or two-way ANOVA Sidak’s multiple comparison test or were assessed with Kruskal–Wallis test between each group and a control. Source data are available for this figure: SourceData FS2.

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